Research Article Activation of Sonic Hedgehog Leads to ...downloads.hindawi.com/journals/bmri/2015/674371.pdf · ophilic meningoencephalitis in humans [ , ]. e infec-tion is acquired

Research ArticleActivation of Sonic Hedgehog Leads to SurvivalEnhancement of Astrocytes via the GRP78-Dependent Pathwayin Mice Infected with Angiostrongylus cantonensis

Kuang-Yao Chen,1 Chien-Ju Cheng,2 and Lian-Chen Wang1,2,3

1Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan2Department of Parasitology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan3Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan

Correspondence should be addressed to Lian-Chen Wang; [email protected]

Received 25 November 2014; Accepted 16 March 2015

Academic Editor: Ruijin Huang

Copyright © 2015 Kuang-Yao Chen et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Angiostrongylus cantonensis infection may cause elevation of ROS and antioxidants in the CSF of infected mice. Astrocytesmay protect the surrounding neurons from oxidative stress-induced cell death by secreting Sonic hedgehog (Shh) via the PI3-K/AKT/Bcl-2 pathway. This study was conducted to determine the role of the Shh signaling pathway in A. cantonensis-infectedBABL/c mice by coculturing astrocytes with living fifth-stage larvae or soluble antigens. The Shh pathway was activated withcorresponding increases in the level of the Shh. Glial fibrillary acidic protein (GFAP) and Shhwere increased in astrocyte coculturedwith living fifth-stage larvae or soluble antigens. The survival of astrocytes pretreated with Shh was significantly elevated incocultures with the antigens but reduced by its inhibitor cyclopamine. The expression of GRP78 and Bcl-2 was significantly higherin astrocytes pretreated with recombinant Shh.These findings suggest that the expression of Shhmay inhibit cell death by activatingBcl-2 through a GRP78-dependent pathway.

1. Introduction

TheHedgehog (Hh) secreted proteins and signaling pathwayplay important roles in animal development [1, 2]. Sonichedgehog (Shh), Desert hedgehog (Dhh), and Indian hedge-hog (Ihh) are the three mammalian homologs that havebeen discovered. Ihh and Dhh are expressed in specifictissues, whereas Shh expression is ubiquitous. Moreover, Shhdeficiency may lead to defects in the embryonic developmentof the neural tube, limbs, and foregut [3].

Shh signaling is mediated by a series of inhibitory steps.In the absence of Shh, the transmembrane Patched (Ptc)receptor blocks the function of the transmembrane proteinSmoothened (Smo). After secretion of Shh, it binds to the Ptcreceptor and Smo is activated.These changes initiate a signal-ing cascade that results in the activation of the transcriptionfactor glioma-associated oncogene-1 (Gli1) [4]. Upon bindingto Shh, Gli1 translocates into the nucleus and controls

the transcription of the target genes shh, ptc, bcl-2, and cyclinD. These changes promote cellular proliferation, cellularsurvival, immune response, and cell fate determination in avariety of organs [5–7].

Activation of the Shh pathway has also been reportedin neurological impairments. This phenomenon has beenattributed to the protection of the central nervous system(CNS) through elevated expression of the antiapoptoticprotein Bcl-2 [8, 9]. Moreover, Shh signaling may protectneurons from damage in Parkinsonism and Alzheimer’sdisease by regulating cell proliferation and apoptosis [10–13].This signaling pathway also promotes blood-brain barrier(BBB) integrity and CNS immune responses. The role of theShh pathway in endogenous immunity at the BBB protectsthe CNS against the entry of proinflammatory lymphocytes[14].

Astrocytes are the most abundant glial cells in thebrain. These cells can reduce H

2O2toxicity by generation

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015, Article ID 674371, 11 pageshttp://dx.doi.org/10.1155/2015/674371

2 BioMed Research International

of antioxidants (glutathione peroxidase) and delay neuronaldeath under H

2O2generation [15]. During brain injury, the

Shh pathway is activated in reactive astrocytes and plays arole in promoting cell proliferation [16, 17]. The secretedshh from activated astrocytes also protects cortical neuronsagainst oxidative stress, suggesting a potential role for Shhin the treatment of brain ischemia and neurodegenerativedisorders [18, 19].

Glucose-regulated protein 78 (GRP78), also called bind-ing immunoglobulin protein (BiP), is an endoplasmic retic-ulum (ER) chaperone of the heat shock protein family [20,21]. Under ER stress, activation of GRP78 may increasecell survival through the unfolded protein response [22]. Inaddition, induction of GRP78 may also protect cells from ERstress-induced apoptosis by activating Bcl-2 and inhibitingBak, Bax, caspase, and CHOP [23, 24]. Under pathologicaldamage conditions, this protein has protective effects ontissues or organs via Bcl-2 activation [25].

Angiostrongylus cantonensis, the rat lungworm, is animportant etiologic agent of eosinophilicmeningitis or eosin-ophilic meningoencephalitis in humans [26, 27]. The infec-tion is acquired by ingesting contaminated snails, slugs, pla-narians, or vegetables, and and thousands of cases of humanangiostrongyliasis have been reported worldwide [28]. Afterinvading the CNS, this parasite induces a wide range ofimmune responses, including eosinophil recruitment andcytokine release (IL-4, IL-5, and eotaxin) via the NF-𝜅Bpathway [29]. In experimentally infected mice, blood-brainbarrier (BBB) dysfunction andCSF eosinophilia always occur[30]. Chung et al. [31, 32] reported that A. cantonensis infec-tion may cause elevation of ROS and antioxidants in the CSFof infected mice. High levels of antioxidants produced byastrocytes may protect the surrounding neurons from oxida-tive stress-induced cell death by inhibiting ROS formation[33].Moreover, astrocytes under oxidative stress are activatedand secrete Shh via the PI3-K/AKT/Bcl-2 pathway [34]. Inthis study, we investigated the activation of the Shh signalingpathway in astrocytes of A. cantonensis-infected mice byimmunohistochemistry and immunofluorescence staining.By coculturing astrocytes with living fifth-stage larvae (L5)or soluble antigens, we determined that activation of Shhsignaling increases the survival rate of astrocytes throughthe GRP78-dependent pathway. These findings may providevaluable insight into the pathological changes during thecourse of cerebral angiostrongyliasis.

2. Materials and Methods

2.1. Parasite and Experimental Infection. Both SD rats andBALB/c mice were purchased from the National LaboratoryAnimal Center, Taipei. In our laboratory, A. cantonensis ismaintained in Biomphalaria glabrata snails and Sprague-Dawley (SD) rats [35]. The life cycle of A. cantonensis wasestablished by isolating L3 fromAchatina fulica snail collectedin Neihu, Taipei, in 1985. After infecting L3 to Sprague-Dawley (SD) rats, the first-stage larvae (L1) were recoveredfrom the rat feces on day 50 after infection and fed to B.glabrata snails. To isolate L3, the infected B. glabrata snails

were killed and the tissues were homogenizedwith an organi-zation homogenizer (Cole-Parmer InstrumentCo., USA) andthen digested with artificial gastric juice (0.6% (w/v) pepsin,pH 2-3) at 37∘C for 45min on day 21 after infection [36]. Eachmouse was inoculated with 25 larvae by stomach intubation.The infected animals were housed separately in plastic cagesand provided with food and drinking water ad libitum. Allprocedures involving animals and their care were reviewedand approved by the Chang Gung University InstitutionalAnimal Care and Use Committee (CGU12-157).

2.2. Preparation of A. cantonensis Soluble Antigens. The L5from A. cantonensis were isolated from the brain tissues ofinfected mice after the mice were anesthetized with 30𝜇LZoletil 50 (Virbac). After washing with saline, phosphatebuffered saline, and distilled water three times, the wormswere disrupted by sonication in lysis buffer (8M urea and 4%CHAPS) containing protease inhibitors (Roche Diagnostics,Basel, Switzerland) on ice for 30 times. The cell lysate wascentrifuged at 10,000×g at 4∘C for 15min, and impuritiesfrom the total cell lysate were removed using the 2-DCleanupKit (GEHealthcare Bio-Science Corp., Piscataway, USA).Theprotein concentration in the preparations was determinedwith the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules,CA, USA) according to the manufacturer’s instructions. Thesoluble antigens were purified from the fifth-stage larvae ofA. cantonensis with lysis buffer and the 2-D Cleanup Kit.These antigens were used to treat the astrocytes, and the cellbiological changes were then observed.

2.3. Cell Culture. Mouse astrocytes (CRL-2535) were pur-chased from the American Type Culture Collection. Dul-becco’s modified Eagle’s medium supplemented with 10%fetal bovine serum and 100U/mL penicillin/streptomycinwas used to maintain the cells throughout the study.The cellswere seeded onto poly-L-lysine coated culture plates at 0.25 ×106 cells/cm2 at 37∘C under 5%CO

2. After culturing for seven

days, the cells grew to a confluent layer of 1-2 × 104 cells/cm2.Over 95% of the cells should be detected by GFAP staining.

2.4. Western Blotting. The levels of the apoptosis-related pro-teins Bax and Bcl-2, GFAP, and Shh were measured by 12.5%SDS-PAGE. A semidry transfer unit (Bio-Rad, Hercules,CA, USA) was used to transfer the proteins from the gelto a nitrocellulose membrane at 0.04mA for 50min. Themembrane was washed with TBS/T three times and thenwith blocking buffer. The primary antibodies were rabbitanti-Shh (1 : 100) (Santa Cruz Biotechnology Inc., USA),rabbit anti-Bax (1 : 500) (Santa Cruz Biotechnology Inc.),rabbit anti-Bcl-2 (1 : 500) (Santa Cruz Biotechnology Inc.),rabbit anti-GFAP (1 : 1,000) (Abcam, UK), rabbit anti-GRP78(1 : 1000) (Sigma-Aldrich), and mouse anti-𝛽-actin (1 : 5000)(Sigma-Aldrich). After incubation with these antibodies at4∘C overnight, the membranes were then washed three timesbefore incubation with the corresponding secondary anti-bodies (goat anti-rabbit IgG 1 : 10,000 and rabbit anti-mouseIgG 1 : 10,000) (Sigma-Aldrich) for 45min. The membraneswere thenwashedwith TBS/T three times and incubatedwith

BioMed Research International 3

Control

14 days

100𝜇m

100𝜇m

50𝜇m

50𝜇m

(a)

0

0.5

1

1.5

Control 7 14 21 28 35 42

(days)

Rela

tive r

atio

Control 7 14 21 28 35 42

GFAP

(days)

𝛽-actin

∗∗∗

(b)

Figure 1: Activation of astrocytes inmice infected withAngiostrongylus cantonensis. (a) A significantly higher GFAP level in the hippocampusof a BALB/c mouse inoculated with 25 third-stage larvae on day 14 after infection was determined by staining with rabbit anti-GFAP. (b)Results from infected mice from day 7 to day 42 after infection were confirmed by Western blotting (𝑛 = 3, ∗𝑃 < 0.05, and ∗∗𝑃 < 0.01).

a mixture of stable peroxide solution (500 𝜇L) and enhancedsolution (500𝜇L) in the dark for 5min. The ImageJ imageanalysis software (http://rsb.info.nih.gov/ij/index.html) wasused to quantitate and compare the concentrations of thetarget proteins (GFAP, Shh, Bax, Bcl-2, and GRP78) and thecontrol (𝛽-actin).

2.5. ELISA. After astrocytes were treatedwith soluble antigenin serum-free DMEM for 0–8 h, the supernatants werecollected at different time points and then centrifuged at500×g for 5min. These specimens were used to detect theconcentration of Shh using a mouse-specific Shh-N ELISAkit (Sigma-Aldrich).

2.6. Immunohistochemistry and Immunofluorescence Staining.The brain tissues from the BALB/c mice were snap-frozen byimmersion in isopentane chilled to −70∘C, and the tissueswere then immediately mounted in OCT medium. Thesamples were stored at −80∘C until sectioning with aMicromOMV cryostat to 10–15 𝜇m. The frozen tissue sections werefixed in 2% (w/v) PFA (paraformaldehyde) andpermeabilizedin 0.5% (v/v) Triton X-100 in PBS before incubation withthe purified antibodies (rabbit anti-Shh (Santa Cruz Biotech-nology Inc., USA, 1 : 50) and rabbit anti-GFAP (Abcam, UK,1 : 500)) for 2 h at room temperature or overnight at 4∘C.Afterincubation, the sections were incubated with the appropri-ate secondary antibodies (DyLight 488-594-conjugate anti-rabbit IgG) (Jackson ImmunoResearch Inc., UK, 1 : 1000) for


(days)

Rela

tive r

atio

(days)

Control 7 14 21 28

Shh

0

0.5

1

2

Control 7 14 21 28

𝛽-actin

1.5

∗∗

(a)

Experimental group

100

x400

x

(b)

Control group

100

x

(c)

Figure 2: Elevation of Shh expression in mice infected with Angiostrongylus cantonensis. (a) Western blotting showing significant increasesin the levels of Shh from day 7 to day 28 after infection (𝑛 = 3, ∗𝑃 < 0.01). (b) An increased Shh expression was demonstrated byimmunofluorescence staining of brain sections from an infectedmouse on day 28. (c) No apparent changes were observed in amouse withoutthe infection on the same day (GFAP: green; Shh: red; colocalization of GFAP and Shh: yellow).

1 h at room temperature. For nuclear counterstaining, thesections were stained with DAPI. The stained sections werethen examined and photographed under a light microscope.

2.7. Cell Viability Assay. Todetermine cell survival, astrocytes(1 × 107 cells/mL) were incubated with 50mL CCK-8 (CellCounting Kit-8) (Sigma-Aldrich) at 37∘C in the dark withmild shaking for 1 h. In the presence of cells, the highlywater-soluble tetrazolium salt WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] produces a water-solubleformazan dye upon reduction. Cell survival is monitored bymeasuring the absorbance of the formazan dye at 450 nmusing a spectrophotometer (Molecular Devices, USA).

2.8. Coculturing and Inhibition Experiments. Before cocul-turing the astrocytes with either L5 or soluble antigens, thecells were incubated in serum-free DMEM for 12 h. Thesecells were then treated with three living L5 (male and female)

for 0–3 h or 500𝜇g soluble antigens for 0–8 h. The groupswere pretreated with either cyclopamine (20𝜇M) (Sigma-Aldrich) or recombinant mouse Sonic hedgehog peptide(Shh) (3 𝜇g) (Sigma-Aldrich) for 1 h and then treated with500𝜇g A. cantonensis soluble antigens for 2 h. Cell viabilitywas measured using the CCK-8 assay (Sigma-Aldrich).

2.9. Apoptosis Assay. A total of 106 astrocytes were harvestedand the pellet was resuspended in 1mL of Annexin V-FITC detection kit reagent (1mL of 1x binding buffer con-taining 10 𝜇L of Annexin V-FITC and 20 𝜇L of propidiumiodide) (Sigma-Aldrich). These cells were incubated at roomtemperature in dark for 10min. Signals were immediatelyanalyzed and quantified using a FASCan flow cytometer (BDBiosciences, USA).

2.10. Statistical Analysis. Student’s 𝑡-test was used to comparethe experimental and control groups. A value of 𝑃 < 0.05wasconsidered statistically significant.


0

4

8

0

50

100

0

50

100

Antigen treatment

Control

M1M1

Cou

nts

0

40

80

120

160

PI

M1M1

200

Cou

nts

0

40

80

120

160

200

Cou

nts

0

40

80

120

160

200

Cou

nts

0

40

80

120

160

200

100 101 102 103 104 100 101 102 103 104

PI100 101 102 103 104

100

101

102

103

104

Annexin V-FITC

Annexin V-FITC

Ann

exin

V-F

ITC

PI100 101 102 103 104 100 101 102 103 104

PI100 101 102 103 104

100

101

102

103

104

Ann

exin

V-F

ITC

Control

Control

ControlAntigen

Antigen

Antigen

∗∗

∗∗

∗∗

Data.003 Data.003 Data.003

Data.002 Data.002 Data.002

(the e

arly

stag

e of a

popt

osis)

Ann

exin

V+

/PI−

cells

rate

(%)

(the l

ate s

tage

of a

popt

osis)

Ann

exin

V+

/PI+

cells

rate

(%)

(nec

rosis

)A

nnex

in V−

/PI+

cells

rate

(%)

Figure 3: Induction of apoptosis in activated astrocytes by culturing with Angiostrongylus cantonensis soluble antigens. Astrocytes weretreated with the 500 𝜇g antigens for 2 h. Cell apoptosis was analyzed by FASCan flow cytometer (Annexin V−/PI−: normal cells, AnnexinV+/PI−: early stage apoptotic cells, Annexin V+/PI+: late stage apoptotic cells, and Annexin V−/PI+: necrosis cells) (𝑛 = 3, ∗∗𝑃 < 0.01).


0

0.5

1

1.5

2

Control 0.5 1 2 3 4 6 8

Control 0.5 1 2 3 4 6 8Re

lativ

e rat

io

(h)

(h)

GFAP

𝛽-actin

∗∗

∗

(a)

0

1

2

3

4

Bax

Rela

tive r

atio

Control 0.5 1 2 3 4 6 8 (h)

Control 0.5 1 2 3 4 6 8 (h)

𝛽-actin

∗∗

(b)

Control 0.5 1 2 3 4 6 8 (h)

Control 0.5 1 2 3 4 6 8 (h)

Shh

𝛽-actin

0

0.5

1

1.5

2

Rela

tive r

atio

∗∗

∗

(c)

0

5

10

0.5 1 2 3 4 6 8Shh

prot

ein

in cu

lture

med

ium

(pg/

mL)

(h)

∗∗

Control

(d)

Figure 4: Induction of Shh release and apoptosis in activated astrocytes by culturing with Angiostrongylus cantonensis soluble antigens.Astrocytes were treated with the 500 𝜇g antigens for 0.5–8 h. Western blotting showed significant increases in the expressions of (a) GFAP,(b) Bax, and (c) Shh in astrocytes after 1 h. (d) The elevation of Shh concentration in the culture medium was confirmed by ELISA (𝑛 = 3,∗𝑃 < 0.05, and ∗∗𝑃 < 0.01).

3. Results

3.1. Activation of Astrocytes. The presence of activated astro-cytes in the brain sections was detected by immunohisto-chemistry. GFAP expression in the astrocytes was signifi-cantly higher in the hippocampus as determined by stainingwith rabbit anti-GFAP on day 14 after infection (Figure 1(a)).Western blotting also showed that GFAP expression wassignificantly higher in the infected group than the controlsfrom day 7 (𝑃 < 0.05) to day 42 (𝑃 < 0.01) after infection(Figure 1(b)).

3.2. Activation of the Shh Pathway and Expression of Shh. Thelevel of Shh was increased in the infected group as deter-mined by Western blotting. The level of the 19-kDa activatedform of Shh was significantly increased beginning on day 7after infection (Figure 2(a)). Immunofluorescence staining ofbrain sections frommice prepared 28 days following infectionwith A. cantonensis showed that the expression of GFAP and

Shh was significantly increased in the cytoplasm of astrocytes(Figures 2(b) and 2(c)).

3.3. Culturing Astrocytes with Soluble Antigens and Worms ofA. cantonensis. Coculturing soluble antigens or living wormsofA. cantonensiswith astrocytes was performed to determinethe induction of Shh release and apoptosis occurred inactivated astrocytes. To assay apoptosis in astrocytes treatedwith A. cantonensis soluble antigens, changes in Annexin V-FITC and propidium iodide were analyzed (Figure 3). In thecontrol group, only 2.07% were early stage apoptotic cells(Annexin V+/PI− cells) whereas the percentage in the cellstreated with 500𝜇g/ml antigens was 49.44%. In addition,4.16% in the control group were late stage apoptotic cells(Annexin V+/PI+ cells) while the corresponding percentagein the experimental group was 43.6%. However, A. cantonen-sis antigens could not induce necrosis in astrocytes (AnnexinV−/PI+) (4.95% in control group versus 0.5% in experimental


Control Antigen treatment

Figure 5: Expression of Shh andGFAP in activated astrocytes by culturing withAngiostrongylus cantonensis soluble antigens. Astrocytes weretreated with the 500 𝜇g antigens for 2 h. Increases in the levels of Shh and GFAP expressions were detected by immunofluorescence stainingof normal and antigen treated astrocytes. Magnification is 1000x (GFAP: green; Shh: red; colocalization of GFAP and Shh: yellow).

group). Therefore, the apoptosis of astrocytes was induced inA. cantonensis infection.

Western blotting showed that the levels of GFAP, Bax, andShh in astrocytes were significantly increased after culturingwith 500𝜇g A. cantonensis soluble antigens for 1 h (𝑃 < 0.01)(Figures 4(a)–4(c)). The level of Shh was also determined byELISA and was significantly elevated in the culture mediumafter 1 h (𝑃 < 0.01) (Figure 4(d)). Immunofluorescencestaining also detected the location of Shh and GFAP in thecytoplasm of astrocytes treated with A. cantonensis solubleantigens (Figure 5). In addition, coculturing the astrocyteswithmale (Figure 6(a)) or female (Figure 6(b))A. cantonensisworms also showed increasing levels of GFAP and Shh after2 h (𝑃 < 0.01).

3.4. Survival Enhancement via the GRP78-Dependent Path-way. ACCK-8 assay was employed to determine the survivalof astrocytes after coculturing experiments with solubleantigens or treatment with a signaling inhibitor. The survivalrates of astrocytes treated with the soluble antigens and Shhwere significantly higher than those treated with the solubleantigens only (𝑃 < 0.01) (Figure 7(a)). However, the survivalrates of astrocytes treated with cyclopamine, a Shh pathwaysignaling inhibitor, were lower than those treated with thesoluble antigens only (𝑃 < 0.01) (Figure 7(b)).The expressionof GRP78 was higher in astrocytes pretreated with Shh (𝑃 <0.01). However, the expression of GRP78 and Bcl-2 was alsohigher in the cultures treated only with the soluble antigens(𝑃 < 0.01). Moreover, the expression of GRP78 and Bcl-2was significantly higher in astrocytes pretreated with therecombinant Shh (Figures 7(c) and 7(d)). These findingssuggest that the expression of Shh may inhibit cell death byactivating Bcl-2 through a GRP78-dependent pathway.

4. Discussion

The rat lungworm A. cantonensis infects the central nervoussystem of humans and causes eosinophilic meningitis oreosinophilic meningoencephalitis [26]. After migration tothe central nervous system through the blood-brain barrier,the third-stage larvae molt twice and develop into fifth-stage larvae. These larvae induce immune responses, such aseosinophil recruitment. In our previous study, we demon-strated that L5 are surrounded by inflammatory cells in theanterior cerebral fissure, hippocampus, posterior cerebralfissure, and cerebellar fissure on day 14 after infection [37].In a previous study, high levels of cathepsin B-like cysteineproteinase 1 and hemoglobinase-type cysteine proteinasewere detected in the excretory-secretory products of A.cantonensis [38].These proteases initialize the degradation ofhost proteins and induce host immune responses by blood-feeding helminthes. They may be important factors thatinduce tissue damage in nonpermissive hosts [39].

The network of blood vessels in the brain is necessaryto provide nutrients, oxygen, and hormones, as well asfor removing carbon dioxide and wastes. The blood-brainbarrier in these blood vessels is formed by endothelialcells and astrocytes and separates the circulating bloodfrom the extracellular fluid in the central nervous system.The BBB only allows the diffusion of small hydrophobicmolecules, such as oxygen and carbon dioxide [40]. Blood-borne pathogens, including the intracellular and extracellularparasites Toxoplasma gondii, Toxocara canis, Trypanosoma,andPlasmodium species, are able to cause blood-brain barrierdysfunction and penetrate into the central nervous system[41–44]. These parasites not only cause damage to the blood-brain barrier but also induce severe immune responses andbrain damage, such as encephalitis, in the host.


0

0.3

0.6

Rela

tive r

atio

0

0.3

0.6

Rela

tive r

atio

Control

GFAP

GFAP

𝛽-actin

∗

∗

Shh

Shh

Control 1 2 3

1 2 3

(h)

(h)

Control 1 2 3 (h)

(a)

GFAP

𝛽-actin

Shh

Control 1 2 3 (h)

0

0.2

0.4

Rela

tive r

atio

Control

∗∗

Shh

1 2 3 (h)

0

0.3

0.6

Rela

tive r

atio

GFAP

∗

Control 1 2 3 (h)

(b)

Figure 6: Induction of astrocyte activation and Shh expression by coculturing astrocytes with Angiostrongylus cantonensis. (a) Male worms.(b) Female worms. The levels of the GFAP and Shh were significantly increased after 2 h (𝑛 = 3, ∗𝑃 < 0.05, and ∗∗𝑃 < 0.01).

After culturing with soluble antigens of A. cantonensis,levels of the apoptosis-related Bax and Bcl-2 were signifi-cantly increased in astrocytes and the activated cells weredetermined to be in the apoptotic state by flow cytom-etry (Figure 3). The elevation of these proteins suggeststhe occurrence of apoptosis. These findings are consistentwith those observed in A. cantonensis-infected mice withmeningoencephalitis [31]. Moreover, the larvae have beenreported to cause severe eosinophilic meningoencephalitis ininfected BALB/c mice.This pathological change in turn leadsto apoptosis and death of the animals [41].

GFAP is the specific marker for astrocytes [45]. In thisstudy, we observed a significant increase in the level of GFAPin the brains of BALB/c mice from day 7 to day 42 day afterinfection, suggesting that the astrocytes were activated afterA. cantonensis infection. Most of the BALB/c mice infectedwith 25 third-stage larvae died around day 28 after infection.

However, someof themmay survive after this time.Therefore,detection of GFAP changes can be extended up to day 42.In in vitro experiments, we also demonstrated apoptosis andactivation in astrocytes induced by A. cantonensis solubleantigens. Although A. cantonensis may cause apoptosis insome astrocytes, the remaining cells were activated by theinfection and showed significant increase in the expression.Moreover, soluble antigens from A. cantonensis can alsoinduce the activation of astrocytes, which in turn causes thesignificant increase in the total level of GFAP (Figure 4(a)).

Inmice infectedwithA. cantonensis, we detected a signifi-cant increase in the expression of Shh.These specific signalingmolecules include Shh, Smo, Patched, and Gli. Moreover,the expression of Shh was significantly elevated in astrocytescultured with either living A. cantonensis worms or solubleantigens. Under oxidative stress, the level of Shh expressedin astrocytes is higher than those in neurons or fibroblasts


0

20

40

60

80

100

120

Control Shh Antigen

∗∗

∗∗

Shh + antigen

CCK-

8re

duct

ion

of as

trocy

tes

(% o

f con

trol)

(a)

Control AntigenCyclopamine

∗∗

∗∗

0

20

40

60

80

100

120

CCK-

8re

duct

ion

of as

trocy

tes

(% o

f con

trol)

antigenCyclopamine +

(b)

GRP78

ControlAntigen

ShhCyclopamine

−

−

−− −

− −

− −−

−−

− − − −+

+

++

++

+ +

Bcl-2

𝛽-actin

(c)

0

0.5

1

1.5

2

Rela

tive r

atio

ControlAntigen

ShhCyclopamine

∗∗

∗∗

−

−

−− −

− −

− −−

−−

− − − −+

+

++

++

+ +

(d)

Figure 7: Activation of the Shh signaling pathway in astrocytes via a GRP78-dependent pathway. Astrocytes were pretreated with (a) Shh(3𝜇g) or (b) cyclopamine (20𝜇M) for 1 h and then treated with soluble Angiostrongylus cantonensis antigens (500 𝜇g) for 2 h. Cell viabilitywas measured using a CCK-8 assay. (c) Detection of GRP78 and Bcl-2 expression during A. cantonensis infection. The cultured astrocyteswere pretreated with Shh (3𝜇g) or cyclopamine (20𝜇M) for 1 h and then treated with the antigens for 2 h. (d) The expression of GRP78 wasdetected with the ImageJ software (𝑛 = 3, ∗𝑃 < 0.05, and ∗∗𝑃 < 0.01).

[19]. During CNS development, Shh signaling is essentialfor neural tube formation [46] and brain development [5,47]. This signaling pathway also promotes BBB integrity andimmunity [14]. In brain injury or ROS generation, Shh playsa protective role by promoting cell proliferation [16, 17]. Theexpression of Shh in activated astrocytes may also protectneurons against oxidative stress [19].

GRP78 is an ER chaperone and may be induced underER stress conditions. It interacts with PERK, ATF6, andIRE1 and induces the inhibition of protein synthesis andthe activation of UPR genes [48]. In addition, GRP78 is animportant antiapoptotic protein because it inhibits BAX andcaspase-7 activation via a GRP78-dependent pathway [49]and by activating Bcl-2 expression [50]. In this study, wedemonstrated the induction of the GRP78/Bcl-2 signalingpathway in mice infected with A. cantonensis. This pathwayis significantly increased during the overexpression of Shhsignaling. These findings suggest that the expression of Shhvia a GRP78-dependent pathway may significantly increase

the survival of astrocytes. In conclusion, the activation of theShh signaling pathway may protect astrocytes by activatingantiapoptotic proteins, such as Bcl-2 and GRP78, after A.cantonensis infection.

In the present study, the role of the Shh signaling path-way in astrocyteswas investigated.Weused the specific inhib-itor cyclopamine to inhibit the Shh pathway and cause celldeath in the presence of soluble antigens. In addition, exog-enous Shh increased the survival of astrocytes.These findingsindicate that the Shh signaling pathway plays a protectiverole to astrocytes in BALB/c mice infected with A. canto-nensis. Moreover, the activation of the Shh signaling pathwayincreased the expression of GRP78 in infected mice.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.


Acknowledgments

This work was supported in part by grants from theNational Science Council, Executive Yuan (NSC101-2320-B-182-045 and NSC102-2320-B-182-028-MY3) and ChangGung Memorial Hospital Research Grants CMRPD1A0112,CMRPD1A0113, and CMRPD1B0352. The authors thank theChang Gung Molecular Medicine Research Center for tech-nical support.

References

[1] J. A. Porter, S. C. Ekker, W.-J. Park et al., “Hedgehog patterningactivity: role of a lipophilic modification mediated by thecarboxy-terminal autoprocessing domain,” Cell, vol. 86, no. 1,pp. 21–34, 1996.

[2] A. P. McMahon, P. W. Ingham, and C. J. Tabin, “Developmentalroles and clinical significance of hedgehog signaling,” CurrentTopics in Developmental Biology, vol. 53, pp. 1–114, 2003.

[3] C. Chiang, Y. Litingtung, E. Lee et al., “Cyclopia and defectiveaxial patterning in mice lacking Sonic hedgehog gene function,”Nature, vol. 383, no. 6599, pp. 407–413, 1996.

[4] K. W. Kinzler, S. H. Bigner, D. D. Bigner et al., “Identification ofan amplified, highly expressed gene in a human glioma,” Science,vol. 236, no. 4797, pp. 70–73, 1987.

[5] J. T. Eggenschwiler andK. V. Anderson, “Cilia and developmen-tal signaling,”Annual Review of Cell andDevelopmental Biology,vol. 23, pp. 345–373, 2007.

[6] O. Nolan-Stevaux, J. Lau, M. L. Truitt et al., “GLI1 is regulatedthrough smoothened-independent mechanisms in neoplasticpancreatic ducts andmediates PDAC cell survival and transfor-mation,”Genes and Development, vol. 23, no. 1, pp. 24–36, 2009.

[7] S. J. Scales and F. J. de Sauvage, “Mechanisms of Hedgehogpathway activation in cancer and implications for therapy,”Trends in Pharmacological Sciences, vol. 30, no. 6, pp. 303–312,2009.

[8] L. Y. Al-Ayadhi, “Relationship between sonic hedgehog protein,brain-derived neurotrophic factor and oxidative stress in autismspectrum Disorders,”Neurochemical Research, vol. 37, no. 2, pp.394–400, 2012.

[9] A. Ghanizadeh, “Malondialdehyde, Bcl-2, superoxide dismu-tase and glutathione peroxidase may mediate the associationof sonic hedgehog protein and oxidative stress in autism,”Neurochemical Research, vol. 37, no. 4, pp. 899–901, 2012.

[10] A. R. Paganelli, O. H. Ocaa, M. I. Prat et al., “The Alzheimer-related gene presenilin-1 facilitates sonic hedgehog expressioninXenopus primary neurogenesis,”Mechanisms ofDevelopment,vol. 107, no. 1-2, pp. 119–131, 2001.

[11] K. Tsuboi and C. W. Shults, “Intrastriatal injection of Sonichedgehog reduces behavioral impairment in a rat model ofParkinson’s disease,” Experimental Neurology, vol. 173, no. 1, pp.95–104, 2002.

[12] M. Bak, C. Hansen, K. F. Henriksen et al., “Mutation analysis ofthe Sonic hedgehog promoter and putative enhancer elementsin Parkinson’s disease patients,” Molecular Brain Research, vol.126, no. 2, pp. 207–211, 2004.

[13] E. M. Torres, C. Monville, P. R. Lowenstein, M. G. Castro, andS. B. Dunnett, “Delivery of sonic hedgehog or glial derivedneurotrophic factor to dopamine-rich grafts in a rat model ofParkinson’s disease using adenoviral vectors: increased yield ofdopamine cells is dependent on embryonic donor age,” BrainResearch Bulletin, vol. 68, no. 1-2, pp. 31–41, 2005.

[14] J. I. Alvarez, A. Dodelet-Devillers, H. Kebir et al., “The hedge-hog pathway promotes blood-brain barrier integrity and CNSimmune quiescence,” Science, vol. 334, no. 6063, pp. 1727–1731,2011.

[15] S. Desagher, J. Glowinski, and J. Premont, “Astrocytes protectneurons from hydrogen peroxide toxicity,” The Journal ofNeuroscience, vol. 16, no. 8, pp. 2553–2562, 1996.

[16] N. M. Amankulor, D. Hambardzumyan, S. M. Pyonteck, O. J.Becher, J. A. Joyce, and E. C. Holland, “Sonic hedgehog pathwayactivation is induced by acute brain injury and regulated byinjury-related inflammation,” Journal of Neuroscience, vol. 29,no. 33, pp. 10299–10308, 2009.

[17] V. M. Heine and D. H. Rowitch, “Hedgehog signaling hasa protective effect in glucocorticoid-induced mouse neonatalbrain injury through an 11𝛽HSD2-dependent mechanism,”TheJournal of Clinical Investigation, vol. 119, no. 2, pp. 267–277, 2009.

[18] E. M. Blanc, A. J. Bruce-Keller, and M. P. Mattson, “Astrocyticgap junctional communication decreases neuronal vulnerabil-ity to oxidative stress-induced disruption of Ca2+ homeostasisand cell death,” Journal ofNeurochemistry, vol. 70, no. 3, pp. 958–970, 1998.

[19] R.-L. Dai, S.-Y. Zhu, Y.-P. Xia et al., “Sonic hedgehog pro-tects cortical neurons against oxidative stress,” NeurochemicalResearch, vol. 36, no. 1, pp. 67–75, 2011.

[20] G. De Ridder, R. Ray, U. K. Misra, and S. V. Pizzo, “Modulationof the unfolded protein response by GRP78 in prostate cancer,”Methods in Enzymology, vol. 489, pp. 245–257, 2011.

[21] R. Kim, M. Emi, K. Tanabe, and S. Murakami, “Role of theunfolded protein response in cell death,”Apoptosis, vol. 11, no. 1,pp. 5–13, 2006.

[22] J. Li and A. S. Lee, “Stress induction of GRP78/BiP and its rolein cancer,” Current Molecular Medicine, vol. 6, no. 1, pp. 45–54,2006.

[23] A. Bruchmann, C. Roller, T. V. Walther et al., “Bcl-2 associatedathanogene 5 (Bag5) is overexpressed in prostate cancer andinhibits ER-stress induced apoptosis,” BMC Cancer, vol. 13,article 96, 2013.

[24] L.-F. Wu, Y.-T. Guo, Q.-H. Zhang et al., “Enhanced antitumoreffects of adenoviral-mediated siRNA against GRP78 geneon adenosine-induced apoptosis in human hepatoma HepG2cells,” International Journal of Molecular Sciences, vol. 15, no. 1,pp. 525–544, 2014.

[25] M. H. Park, Y. K. Lee, Y. H. Lee et al., “Chemokines releasedfrom astrocytes promote chemokine receptor 5-mediated neu-ronal cell differentiation,” Experimental Cell Research, vol. 315,no. 16, pp. 2715–2726, 2009.

[26] L.-C. Wang, I.-H. Wang, and J.-R. Jou, “Optic neuritissecondary to Angiostrongylus cantonensis infection,” OcularImmunology and Inflammation, vol. 14, no. 3, pp. 189–191, 2006.

[27] N. S. Hochberg, S. Y. Park, B. G. Blackburn et al., “Distributionof eosinophilic meningitis cases attributable to Angiostrongyluscantonensis, Hawaii,” Emerging Infectious Diseases, vol. 13, no. 11,pp. 1675–1680, 2007.

[28] Q.-P. Wang, Z.-D. Wu, J. Wei, R. L. Owen, and Z.-R. Lun,“Human angiostrongylus cantonensis: an update,” EuropeanJournal of Clinical Microbiology and Infectious Diseases, vol. 31,no. 4, pp. 389–395, 2012.

[29] K. P. Lan, C. J. Wang, J. D. Hsu, K. M. Chen, S. C. Lai, and H. H.Lee, “Induced eosinophilia and proliferation in Angiostrongyluscantonensis-infectedmouse brain are associatedwith the induc-tion of JAK/STAT1, IAP/NF-kappaB and MEKK1/JNK signals,”Journal of Helminthology, vol. 78, no. 4, pp. 311–317, 2004.


[30] H.-C. Tsai, Y.-L. Huang, Y.-C. Liu et al., “Dynamic changesof hepatocyte growth factor in eosinophilic meningitis causedby Angiostrongylus cantonensis infection,” American Journal ofTropicalMedicine andHygiene, vol. 80, no. 6, pp. 980–982, 2009.

[31] L.-Y. Chung, C.-H. Chen, L.-C. Wang, S.-J. Chang, and C.-M.Yen, “Oxidative stress in mice infected with Angiostrongyluscantonensis coincides with enhanced glutathione-dependentenzymes activity,” Experimental Parasitology, vol. 126, no. 2, pp.178–183, 2010.

[32] L.-Y. Chung, L.-C.Wang, C.-H. Chen,H.-Y. Lin, andC.-M. Yen,“Kinetic change of oxidative stress in cerebrospinal fluid ofmiceinfected with Angiostrongylus cantonensis,” Redox Report, vol.15, no. 1, pp. 43–48, 2010.

[33] R. Dringen, J. M. Gutterer, and J. Hirrlinger, “Glutathionemetabolism in brain: metabolic interaction between astrocytesand neurons in the defense against reactive oxygen species,”European Journal of Biochemistry, vol. 267, no. 16, pp. 4912–4916,2000.

[34] Y.-P. Xia, R.-L. Dai, Y.-N. Li et al., “The protective effect ofsonic hedgehog is mediated by the propidium iodide 3-kinase/AKT/Bcl-2 pathway in cultured rat astrocytes under oxidativestress,” Neuroscience, vol. 209, pp. 1–11, 2012.

[35] L.-C. Wang, D. Chao, and E.-R. Chen, “Acquired immunity inrats against Angiostrongylus cantonensis infection,” Internatio-nal Journal for Parasitology, vol. 19, no. 6, pp. 617–620, 1989.

[36] L.-C. Wang, D. Chao, and E.-R. Chen, “Experimental infec-tion routes of Angiostrongylus cantonensis in mice,” Journal ofHelminthology, vol. 65, no. 4, pp. 296–300, 1991.

[37] L.-C. Wang, D.-P. Wan, S.-M. Jung, C.-C. Chen, H.-F. Wong,and Y.-L. Wan, “Magnetic resonance imaging findings in thebrains of rabbits infected with Angiostrongylus cantonensis: along-term investigation,” Journal of Parasitology, vol. 91, no. 5,pp. 1237–1239, 2005.

[38] A. L. Morassutti, K. Levert, P. M. Pinto, A. J. da Silva, P.Wilkins,and C. Graeff-Teixeira, “Characterization of Angiostrongyluscantonensis excretory-secretory proteins as potential diagnostictargets,” Experimental Parasitology, vol. 130, no. 1, pp. 26–31,2012.

[39] K.-P. Hwang, S.-H. Chang, and L.-C. Wang, “Alterations in theexpression level of a putative aspartic protease in the develop-ment of Angiostrongylus cantonensis,” Acta Tropica, vol. 113, no.3, pp. 289–294, 2010.

[40] L. Renia, S. W. Howland, C. Claser et al., “Cerebral malaria:mysteries at the blood-brain barrier,”Virulence, vol. 3, no. 2, pp.193–201, 2012.

[41] K.-M. Chen, H.-H. Lee, S.-C. Lai, L.-S. Hsu, C.-J. Wang, andJ.-Y. Liu, “Apoptosis in meningoencephalitis of Angiostrongyluscantonensis-infected mice,” Experimental Parasitology, vol. 119,no. 3, pp. 385–390, 2008.

[42] C.M.Hamilton, S. Brandes, C. V.Holland, and E. Pinelli, “Cyto-kine expression in the brains of Toxocara canis-infected mice,”Parasite Immunology, vol. 30, no. 3, pp. 181–185, 2008.

[43] S. M. Feustel, M. Meissner, and O. Liesenfeld, “Toxoplasmagondii and the blood-brain barrier,” Virulence, vol. 3, no. 2, pp.182–192, 2012.

[44] W.Masocha andK. Kristensson, “Passage of parasites across theblood-brain barrier,” Virulence, vol. 3, no. 2, pp. 202–212, 2012.

[45] M. Salouci, N. Antoine, M. K. Shikh Al Sook et al., “Develop-mental profiles of GFAP-positive astrocytes in sheep cerebel-lum,” Veterinary Research Communications, vol. 38, no. 4, pp.279–285, 2014.

[46] J. Lee, K. A. Platt, P. Censullo, and A. Ruiz i Altaba, “Gli1 is atarget of Sonic hedgehog that induces ventral neural tubedevelopment,”Development, vol. 124, no. 13, pp. 2537–2552, 1997.

[47] N. Dahmane, P. Sanchez, Y. Gitton et al., “The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis,”Development, vol. 128, no. 24, pp. 5201–5212, 2001.

[48] Y. Jiang, H. Lv, M. Liao et al., “GRP78 counteracts cell deathand protein aggregation caused bymutant huntingtin proteins,”Neuroscience Letters, vol. 516, no. 2, pp. 182–187, 2012.

[49] E. Lee, P. Nichols, S. Groshen, D. Spicer, and A. S. Lee, “GRP78as potential predictor for breast cancer response to adjuvanttaxane therapy,” International Journal of Cancer, vol. 128, no. 3,pp. 726–731, 2011.

[50] H. Zhou, Y. Zhang, Y. Fu, L. Chan, and A. S. Lee, “Novel mech-anism of anti-apoptotic function of 78-kDa glucose-regulatedprotein (GRP78): endocrine resistance factor in breast cancer,through release of B-cell lymphoma 2 (BCL-2) from BCL-2-interacting killer (BIK),”TheJournal of Biological Chemistry, vol.286, no. 29, pp. 25687–25696, 2011.

Submit your manuscripts athttp://www.hindawi.com

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation http://www.hindawi.com

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation http://www.hindawi.com Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttp://www.hindawi.com Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttp://www.hindawi.com

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research


International Journal of

Microbiology

Documents

Research Article Activation of Sonic Hedgehog Leads to ...downloads.hindawi.com/journals/bmri/2015/674371.pdf · ophilic meningoencephalitis in humans [ , ]. e infec-tion is acquired